The present invention relates to a novel high throughput assay for monitoring polyion (polycation or polyanion) degradation or polymerization and for determining a molecular weight of a polyion. More particularly, the present invention relates to a high throughput assay for monitoring the activity of enzymes which either degrade or synthesize polyions and for screening for potential modulators (inhibitors or activators) of such enzymes, however, physical and chemical degradation/polymerization of polyions and modulators thereof can also be monitored by the method of the present invention. Most, particularly, the present invention relates to a high throughput assay for monitoring the catalytic activity of glycosaminoglycans (GAGs) degrading enzymes and for screening of modulators, especially inhibitors, thereof.
Proteoglycans (PGs):
Proteoglycans (previously named mucopolysaccharides) are remarkably complex molecules and are found in every tissue of the body. They are associated with each other and also with other major structural components, such as collagen and elastin. Some PGs interact with certain adhesive proteins, such as fibronectin and laminin.
Glycosaminoglycans (GAGs):
Glycosaminoglycans (GAGs) proteoglycans are polyanions and hence bind polycations and cations, such as Na+ and K+. This latter ability attracts water by osmotic pressure into the extracellular matrix and contributes to its turgor. GAGs also gel at relatively low concentrations. The long extended nature of the polysaccharide chains of GAGs and their ability to gel, allow relatively free diffusion of small molecules, but restrict the passage of large macromolecules. Because of their extended structures and the huge macromolecular aggregates they often form, they occupy a large volume of the extracellular matrix relative to proteins. Murry R K and Keeley F W; Harper""s Biochemistry, 24th Ed. Ch. 57. pp. 667-85.
Heparan sulfate (HS) proteoglycans:
Heparan sulfate (HS) proteoglycans are acidic polysaccharide-protein conjugates associated with cell membranes and extracellular matrices. They bind avidly to a variety of biologic effector molecules, including extracellular matrix components, growth factor, growth factor binding proteins, cytokines, cell adhesion molecules, proteins of lipid metabolism, degradative enzymes, and protease inhibitors. Owing to these interactions, heparan sulfate proteoglycans play a dynamic role in biology, in fact most functions of the proteoglycans are attributable to the heparan sulfate chains, contributing to cell-cell interactions and cell growth and differentiation in a number of systems. It maintains tissue integrity and endothelial cell function. It serves as an adhesion molecule and presents adhesion-inducing cytokines (especially chemokines), facilitating localization and activation of leukocytes. The adhesive effect of heparan sulfate-bound chemokines can be abrogated by exposing the extracellular matrices to heparanase before or after the addition of chemokines. Heparan sulfate modulates the activation and the action of enzymes secreted by inflammatory cells. The function of heparan sulfate changes during the course of the immune response are due to changes in the metabolism of heparan sulfate and to the differential expression of and competition between heparan sulfate-binding molecules. Selvan R S et al.; Ann. NY Acad. Sci. 1996; 797:127-139.
Other PGs and GAGs, such as hyaluronic acid, chondroitin sulfates, keratan sulfates I, II, dermatan sulfate and heparin have also important physiological functions.
GAG degrading enzymes:
Degradation of GAGs is carried out by a battery of lysosomal hydrolases. These include certain endoglycosidases, such as, but not limited to, mammal heparanase (U.S. Pat. No. 5,968,822 for recombinant and WO91/02977 for native human heparanase) and connective tissue activating peptide III (CTAP, WO95/04158 for native and U.S. Pat. No. 4,897,348 for recombinant CTAP) which degrade heparan sulfate and to a lesser extent heparin; heparinase I, II and III (U.S. Pat No. 5,389,539 for the native form and WO95/34635 A1, U.S. Pat. No. 5,714,376 and U.S. Pat. No. 5,681,733 for the recombinant form), e.g., from Flavobacterium heparinum and Bacillus sp., which cleave heparin-like molecules; heparitinase T-I, T-II, T-III and T-VI from Bacillus circulans (U.S. Pat. No. 5,405,759, JO 4278087 and JP04-278087); xcex2-glucoronidase; chondroitinase ABC (EC 4.2.2.4) from Proteus vulgaris, AC (EC 4.2.2.5) from Arthrobacter aurescens or Flavobacterium heparinum, B and C (EC 4.2.2) from Flavobacterium heparinum which degrade chondroitin sulfate; hyaluronidase from sheep or bovine testes which degrade hyaluronidase and chondroitin sulfate; various exoglycosidases (e.g., xcex2-glucoronidase EC 3.2.1.31) from bovine liver, mollusks and various bacteria; and sulfatases (e.g., iduronate sulfatase) EC 3.1.6.1 from limpets (Patella vulgaris), Aerobacter aerogens, Abalone entrails and Helix pomatia, generally acting in sequence to degrade the various GAGs.
Heparanase:
One important enzyme involved in the catabolism of certain GAGs is heparanase. It is an endo-xcex2-glucuronidase that cleaves heparan sulfate at specific interchain sites. Interaction of T and B lymphocytes, platelets, granulocytes, macrophages and mast cells with the subendothelial extracellular matrix (ECM) is associated with degradation of heparan sulfate by heparanase activity. The enzyme is released from intracellular compartments (e.g., lysosomes or specific granules) in response to various activation signals (e.g., thrombin, calcium ionophore, immune complexes, antigens and mitogens), suggesting its regulated involvement in inflammation and cellular immunity. Vlodavsky I et al.; Invasion Metas. 1992; 12(2):112-27.
Cloning and expression of the heparanase gene:
A purified fraction of heparanase isolated from human hepatoma cells was subjected to tryptic digestion. Peptides were separated by high pressure liquid chromatography and micro sequenced. The sequence of one of the peptides was used to screen data bases for homology to the corresponding back translated DNA sequence. This procedure led to the identification of a clone containing an insert of 1020 base pairs (bp) which included an open reading frame of 963 bp followed by 27 bp of 3xe2x80x2 untranslated region and a poly A tail. The new gene was designated hpa. Cloning of the missing 5xe2x80x2 end of hpa was performed by PCR amplification of DNA from placenta cDNA composite. The entire heparanase cDNA was designated phpa. The joined cDNA fragment contained an open reading frame which encodes a polypeptide of 543 amino acids with a calculated molecular weight of 61,192 daltons. Cloning an extended 5xe2x80x2 sequence was enabled from the human SK-hep1 cell line by PCR amplification using the Marathon RACE system. The 5xe2x80x2 extended sequence of the SK-hep1 hpa cDNA was assembled with the sequence of the hpa cDNA isolated from human placenta. The assembled sequence contained an open reading frame which encodes a polypeptide of 592 amino acids with a calculated molecular weight of 66,407 daltons. The cloning procedures are described in length in U.S. Pat. No. 5,968,822, PCT Application No. U.S. Ser. No. 98/17954 and U.S. patent application Ser. Nos. 09/109,386 now abandoned and 09/258,892 now abandoned.
The ability of the hpa gene product to catalyze degradation of heparan sulfate (HS) in vitro was examined by expressing the entire open reading frame of hpa in High five and Sf21 insect cells, and the mammalian human 293 embryonic kidney cell line expression systems. Extracts of infected cells were assayed for heparanase catalytic activity. For this purpose, cell lysates were incubated with sulfate labeled, ECM-derived HSPG (peak I), followed by gel filtration analysis (Sepharose 6B) of the reaction mixture. While the substrate alone consisted of high molecular weight material, incubation of the HSPG substrate with lysates of cells infected with hpa containing virus resulted in a complete conversion of the high molecular weight substrate into low molecular weight labeled heparan sulfate degradation fragments (see, for example, U.S. patent application Ser. No. 09/260,038 now U.S. Pat. No. 6,348,344).
In subsequent experiments, the labeled HSPG substrate was incubated with the culture medium of infected High Five and Sf21 cells. Heparanase catalytic activity, reflected by the conversion of the high molecular weight HSPG substrate into low molecular weight HS degradation fragments, was found in the culture medium of cells infected with the pFhpa virus, but not the control pF1 virus.
Altogether, these results indicate that the heparanase enzyme is expressed in an active form by cells infected with Baculovirus or mammalian expression vectors containing the newly identified human hpa gene.
In other experiments, it was demonstrated that the heparanase enzyme expressed by cells infected with the pFhpa virus is capable of degrading HS complexed to other macromolecular constituents (e.g., fibronectin, laminin, collagen) present in a naturally produced intact ECM (Ser. No. 09/260,038 now U.S. Pat. No. 6,348,344), in a manner similar to that reported for highly metastatic tumor cells or activated cells of the immune system (Vlodavsky, I., Eldor, A., Haimovitz-Friedman, A., Matzner, Y., Ishai-Michaeli, R., Levi, E., Bashkin, P., Lider, O., Naparstek, Y., Cohen, I. R., and Fuks, Z. (1992) Expression of heparanase by platelets and circulating cells of the immune system: Possible involvement in diapedesis and extravasation. Invasion and Metastasis, 12, 112-127; Vlodavsky, I., Mohsen, M., Lider, O., Ishai-Michaeli, R., Ekre, H. -P., Svahn, C. M., Vigoda, M., and Peretz, T. (1995). Inhibition of tumor metastasis by heparanase inhibiting species of heparin. Invasion and Metastasis, 14: 290-302).
Purification of the recombinant heparanase enzyme:
Sf21 insect cells were infected with pFhpa virus and the culture medium was applied onto a heparin-Sepharose column. Fractions were eluted with a salt gradient (0.35-2.0 M NaCl) and tested for heparanase catalytic activity and protein profile (SDS/PAGE followed by silver staining). Heparanase catalytic activity correlated with the appearance of a about 63 kDa protein band in fractions 19-24, consistent with the expected molecular weight of the hpa gene product. Active fractions eluted from heparin-Sepharose were pooled, concentrated and applied onto a Superdex 75 FPLC gel filtration column. Aliquots of each fraction were tested for heparanase catalytic activity and protein profile. A correlation was found between the appearance of a major protein (approximate molecular weight of 63 kDa) in fractions 4-7 and heparanase catalytic activity. This protein was not present in medium conditioned by control non-infected Sf21 cells subjected to the same purification protocol. Recently, an additional purification protocol was applied, using a single step chromatography with source-S ion exchange column. This purification resulted in a purified protein to a degree of 90%. Further details concerning these purification procedures are disclosed in U.S. patent application Ser. Nos. 09/260,038 now U.S. Pat. No. 6,348,344 and 09/071,618 now abandoned, both are incorporated by reference as if fully set forth herein.
Involvement of heparanase in tumor cell invasion and metastasis:
Circulating tumor cells arrested in the capillary beds of different organs must invade the endothelial cell lining and degrade its underlying basement membrane (BM) in order to escape into the extravascular tissue(s) where they establish metastasis (Liotta, L. A., Rao, C. N., and Barsky, S. H. (1983). Tumor invasion and the extracellular matrix. Lab. Invest., 49, 639-649). Several cellular enzymes (e.g., collagenase IV, plasminogen activator, cathepsin B, elastase) are thought to be involved in degradation of the BM (Liotta, L. A., Rao, C. N., and Barsky, S. H. (1983). Tumor invasion and the extracellular matrix. Lab. Invest., 49, 639-649). Among these enzymes is an endo-xcex2-D-glucuronidase (heparanase) that cleaves HS at specific intrachain sites (Vlodavsky, I., Eldor, A., Haimovitz-Friedman, A., Matzner, Y., Ishai-Michaeli, R., Levi, E., Bashkin, P., Lider, O., Naparstek, Y., Cohen, I. R., and Fuks, Z. (1992). Expression of heparanase by platelets and circulating cells of the immune system: Possible involvement in diapedesis and extravasation. Invasion and Metastasis, 12, 112-127; Nakajima, M., Irimura, T., and Nicolson, G. L. (1988). Heparanase and tumor metastasis. J. Cell. Biochem., 36, 157-167; Vlodavsky, I., Fuks, Z., Bar-Ner, M., Ariav, Y., and Schirrmacher, V. (1983). Lymphoma cell mediated degradation of sulfated proteoglycans in the subendothelial extracellular matrix: Relationship to tumor cell metastasis. Cancer Res., 43, 2704-2711; Vlodavsky, I., Ishai-Michaeli, R., Bar-Ner, M., Fridman, R., Horowitz, A. T., Fuks, Z. and Biran, S. Involvement of heparanase in tumor metastasis and angiogenesis. Is. J. Med. 24:464-470, 1988). Expression of a HS degrading heparanase was found to correlate with the metastatic potential at mouse lymphoma (Vlodavsky, I., Fuks, Z., Bar-Ner, M., Ariav, Y., and Schirrmacher, V. (1983). Lymphoma cell mediated degradation of sulfated proteoglycans in the subendothelial extracellular matrix: Relationship to tumor cell metastasis. Cancer Res., 43, 2704-2711), fibrosarcoma and melanoma (Nakajima, M., Irimura, T., and Nicolson, G. L. (1988). Heparanase and tumor metastasis. J. Cell. Biochem., 36, 157-167) cells. The same is true for human breast, bladder and prostate carcinoma cells (see U.S. patent application Ser. No. 09/109,386 now abandoned, which is incorporated by reference as if fully set forth herein). Moreover, elevated levels of heparanase were detected in sera (Nakajima, M., Irimura, T., and Nicolson, G. L. (1988). Heparanase and tumor metastasis. J. Cell. Biochem., 36, 157-167) and urine (U.S. patent application Ser. No. 09/109,386 now abandoned) of metastatic tumor bearing animals and cancer patients and in tumor biopsies (Vlodavsky, I., Ishai-Michaeli, R., Bar-Ner, M., Fridman, R., Horowitz, A. T., Fuks, Z. and Biran, S. Involvement of heparanase in tumor metastasis and angiogenesis. Is. J. Med. 24:464-470, 1988). Treatment of experimental animals with heparanase alternative substrates and inhibitor (e.g., non-anticoagulant species of low molecular weight heparin, laminarin sulfate) markedly reduced ( greater than 90%) the incidence of lung metastases induced by B16 melanoma, Lewis lung carcinoma and mammary adenocarcinoma cells (Vlodavsky, I., Mohsen, M., Lider, O., Ishai-Michaeli, R., Ekre, H. -P., Svahn, C. M., Vigoda, M., and Peretz, T. (1995). Inhibition of tumor metastasis by heparanase inhibiting species of heparin. Invasion and Metastasis, 14: 290-302; Nakajima, M., Irimura, T., and Nicolson, G. L. (1988). Heparanase and tumor metastasis. J. Cell. Biochem., 36, 157-167; Parish, C. R., Coombe, D. R., Jakobsen, K. B., and Underwood, P. A. (1987). Evidence that sulfated polysaccharides inhibit tumor metastasis by blocking tumor cell-derived heparanase. Int. J. Cancer, 40, 511-517), indicating that heparanase inhibitors may be applied to inhibit tumor cell invasion and metastasis.
The studies on the control of tumor progression by its local environment, focus on the interaction of cells with the extracellular matrix (ECM) produced by cultured corneal and vascular endothelial cells (EC) (Vlodavsky, I., Liu, G. M., and Gospodarowicz, D. (1980). Morphological appearance, growth behavior and migratory activity of human tumor cells maintained on extracellular matrix vs. plastic. Cell, 19, 607-616; Vlodavsky, I., Bar-Shavit, R., Ishai-Michaeli, R., Bashkin, P., and Fuks, Z. (1991). Extracellular sequestration and release of fibroblast growth factor: a regulatory mechanism? Trends Biochem. Sci., 16, 268-271). This ECM closely resembles the subendothelium in vivo in its morphological appearance and molecular composition. It contains collagens (mostly type III and IV, with smaller amounts of types I and V), proteoglycans (mostly heparan sulfate- and dermatan sulfate-proteoglycans, with smaller amounts of chondroitin sulfate proteoglycans), laminin, fibronectin, entactin and elastin (Parish, C. R., Coombe, D. R., Jakobsen, K. B., and Underwood, P. A. (1987). Evidence that sulfated polysaccharides inhibit tumor metastasis by blocking tumor cell-derived heparanase. Int. J. Cancer, 40, 511-517; Vlodavsky, I., Liu, G. M., and Gospodarowicz, D. (1980). Morphological appearance, growth behavior and migratory activity of human tumor cells maintained on extracellular matrix vs. plastic. Cell, 19, 607-616). The ability of cells to degrade HS in the ECM was studied by allowing cells to interact with a metabolically sulfate labeled ECM, followed by gel filtration (Sepharose 6B) analysis of degradation products released into the culture medium (Vlodavsky, I., Fuks, Z., Bar-Ner, M., Ariav, Y., and Schirrmacher, V. (1983). Lymphoma cell mediated degradation of sulfated proteoglycans in the subendothelial extracellular matrix: Relationship to tumor cell metastasis. Cancer Res., 43, 2704-2711). While intact HSPG are eluted next to the void volume of the column (Kav less than 0.2, Mr of about 0.5xc3x97106), labeled degradation fragments of HS side chains are eluted more toward the Vt of the column (0.5 less than kav less than 0.8, Mr of about 5-7xc3x97103) (Vlodavsky, I., Fuks, Z., Bar-Ner, M., Ariav, Y., and Schirrmacher, V. (1983). Lymphoma cell mediated degradation of sulfated proteoglycans in the subendothelial extracellular matrix: Relationship to tumor cell metastasis. Cancer Res., 43, 2704-2711). Compounds which efficiently inhibit the ability of heparanase to degrade the above-described naturally produced basement membrane-like substrate, were also found to inhibit experimental metastasis in mice and rats (Vlodavsky, I., Mohsen, M., Lider, O., Ishai-Michaeli, R., Ekre, H. -P., Svahn, C. M., Vigoda, M., and Peretz, T. (1995). Inhibition of tumor metastasis by heparanase inhibiting species of heparin. Invasion and Metastasis, 14: 290-302; Nakajima, M., Irimura, T., and Nicolson, G. L. (1988). Heparanase and tumor metastasis. J. Cell. Biochem., 36, 157-167; Parish, C. R., Coombe, D. R., Jakobsen, K. B., and Underwood, P. A. (1987). Evidence that sulfated polysaccharides inhibit tumor metastasis by blocking tumor cell-derived heparanase. Int. J. Cancer, 40, 511-517; Coombe D R, Parish C R, Ramshaw I A, Snowden J M: Analysis of the inhibition of tumor metastasis by sulfated polysaccharides. Int J Cancer 1987; 39:82-8). A reliable in vitro screening system for heparanase inhibiting compounds may hence be applied to identify and develop potent anti-metastatic drugs.
Possible involvement of heparanase in tumor angiogenesis:
It was previously demonstrated that heparanase may not only function in cell migration and invasion, but may also elicit an indirect neovascular response (Vlodavsky, I., Bar-Shavit, R., Ishai-Michaeli, R., Bashkin, P., and Fuks, Z. (1991). Extracellular sequestration and release of fibroblast growth factor: a regulatory mechanism? Trends Biochem. Sci., 16, 268-271). The results suggest that the ECM HSPGs provide a natural storage depot for xcex2FGF and possibly other heparin-binding growth promoting factors. Heparanase mediated release of active xcex2FGF from its storage within ECM may therefore provide a novel mechanism for induction of neovascularization in normal and pathological situations (Vlodavsky, I., Bar-Shavit, R., Korner, G., and Fuks, Z. (1993). Extracellular matrix-bound growth factors, enzymes and plasma proteins. In Basement membranes: Cellular and molecular aspects (eds. D. H. Rohrbach and R. Timpl), pp 327-343. Academic press Inc., Orlando, Fla.; Thunberg L, Backstrom G, Grundberg H, Risenfield J, Lindahl U: Themolecular size of the antithrombin-binding sequence in heparin. FEBS Lett 1980; 117:203-206).
Expression of heparanase by cells of the immune system:
Heparanase catalytic activity correlates with the ability of activated cells of the immune system to leave the circulation and elicit both inflammatory and autoimmune responses. Interaction of platelets, granulocytes, T and B lymphocytes, macrophages and mast cells with the subendothelial ECM is associated with degradation of heparan sulfate (HS) by heparanase catalytic activity (Vlodavsky, I., Eldor, A., Haimovitz-Friedman, A., Matzner, Y., Ishai-Michaeli, R., Levi, E., Bashkin, P., Lider, O., Naparstek, Y., Cohen, I. R., and Fuks, Z. (1992). Expression of heparanase by platelets and circulating cells of the immune system: Possible involvement in diapedesis and extravasation. Invasion and Metastasis, 12, 112-127). The enzyme is released from intracellular compartments (e.g., lysosomes, specific granules) in response to various activation signals (e.g., thrombin, calcium ionophore, immune complexes, antigens, mitogens), suggesting its regulated involvement and presence in inflammatory sites and autoimmune lesions. Heparan sulfate degrading enzymes released by platelets and macrophages are likely to be present in atherosclerotic lesions (Campbell, K. H., Rennick, R. E., Kalevich, S. G., and Campbell, G. R. (1992) Exp. Cell Res. 200, 156-167). Treatment of experimental animals with heparanase alternative substrates (e.g., non-anticoagulant species of low molecular weight heparin) markedly reduced the incidence of experimental autoimmune encephalomyelitis (EAE), adjuvant arthritis and graft rejection (Vlodavsky, I., Eldor, A., Haimovitz-Friedman, A., Matzner, Y., Ishai-Michaeli, R., Levi, E., Bashkin, P., Lider, O., Naparstek, Y., Cohen, I. R., and Fuks, Z. (1992). Expression of heparanase by platelets and circulating cells of the immune system: Possible involvement in diapedesis and extravasation. Invasion and Metastasis, 12, 112-127; Lider, O., Baharav, E., Mekori, Y., Miller, T., Naparstek, Y., Vlodavsky, I. and Cohen, I. R. Suppression of experimental autoimmune diseases and prolongation of allograft survival by treatment of animals with heparinoid inhibitors of T lymphocyte heparanase. J. Clin. Invest. 83:752-756, 1989) in experimental animals, indicating that heparanase inhibitors may be applied to inhibit autoimmune and inflammatory diseases (Vlodavsky, I., Eldor, A., Haimovitz-Friedman, A., Matzner, Y., Ishai-Michaeli, R., Levi, E., Bashkin, P., Lider, O., Naparstek, Y., Cohen, I. R., and Fuks, Z. (1992). Expression of heparanase by platelets and circulating cells of the immune system: Possible involvement in diapedesis and extravasation. Invasion and Metastasis, 12, 112-127; Lider, O., Baharav, E., Mekori, Y., Miller, T., Naparstek, Y., Vlodavsky, I. and Cohen, I. R. Suppression of experimental autoimmune diseases and prolongation of allograft survival by treatment of animals with heparinoid inhibitors of T lymphocyte heparanase. J. Clin. Invest. 83:752-756, 1989). A reliable in vitro screening system for heparanase inhibiting compounds may hence be applied to identify and develop non-toxic anti-inflammatory drugs for the treatment of multiple sclerosis and other inflammatory diseases.
Recombinant heparanase for screening purposes:
Research aimed at identifying and developing inhibitors of heparanase catalytic activity has been handicapped by the lack of a consistent and constant source of a purified and highly active heparanase enzyme and of a reliable screening system. The recent cloning, expression and purification of the human heparanase-encoding gene offer, for the first time, a most appropriate and reliable source of active recombinant enzyme for screening of anti-heparanase antibodies and small compounds which may inhibit the enzyme and hence be applied to identify and develop drugs that may inhibit tumor metastasis, autoimmune and inflammatory diseases.
Screening for specific inhibitors using a combinatorial library:
A new approach aimed at rational drug discovery was recently developed for screening for specific biological activities. According to the new approach, a large library of chemically diversed molecules are screened for the desired biological activity. The new approach has become an effective and hence important tool for discovery of new drugs. The new approach is based on xe2x80x9ccombinatorialxe2x80x9d synthesis of a diverse set of molecules in which several components predicted to be associated with the desired biological activity are systematically varied. The advantage of a combinatorial library over the alternative use of natural extracts for screening for desired biologically active compounds is that all the components comprising the library are known in advance (Farndale R. W., Sayers C. A., Barrett A. J. A Direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures. Connective Tissue Res. 1990; 24: 267-275).
In combinatorial screening, the number of hits discovered is proportional to the number of molecules tested. This is true even when knowledge concerning the target is unavailable. The large number of compounds, which may reach thousands of compounds tested per day, can only be screened, provided that a suitable assay involving a high throughput screening technique, in which laboratory automation and robotics may be applied, exists.
Prior art heparanase catalytic activity assays:
Several methods for determining heparanase catalytic activity have been developed throughout the years. Many of the different methods are based on radiolabeling of a substrate (either in vitro or metabolically, as described above) and analysis of its degradation products released due to heparanase catalytic activity. These prior art methods suffer several disadvantages and limitations as follows.
First, the measurement of catalytic activity is qualitative and not quantitative. This is due to the following reasons (i) the radioactive labeling is not spread evenly along the substrate chain, therefore, radioactivity may not correlate precisely with activity; (ii) since heparanase substrates are long substrate chains, a released product can be, in fact, a substrate of heparanase, however while executing any of the prior art methods, cleavage events of released products are not monitorable. Moreover, multiple cleavage events of small portions of the substrate chain are indistinguishable from fewer cleavage events, yet of longer substrate chains. Thus, not all, and in many cases, depending on the substrate chain length, not even most, of the cleavage events catalyzed by the enzyme are detectable, thereby affecting the linearity of the assay.
Second, these prior art methods are cumbersome, time-consuming and do not allow activity determination of a large number of samples simultaneously. In most cases, both preparation of the radiolabeled substrate and separation of the degradation products from the uncleaved substrate involve long and complex procedures and handling with radioactive material which calls strict safety procedures.
Third, these prior art methods for determining heparanase catalytic activity involve modification of the substrate by either iodination at glucosamine residues, or either O- or N-acetylation of the partially de-N-sulfated substrate. Such procedures may result in masking heparanase cleavage sites, or alternatively creating new heparanase sites.
These different prior art methods also have specific disadvantages specifically associated with each of which. Some methods involve biosynthetic radiolabeling of ECM associated HSPG and detection of HS chain degradation by gel filtration analysis of the radiolabeled material released from the labeled ECM (Vlodavsky, I., Eldor, A., Haimovitz-Friedman, A., Matzner, Y., Ishai-Michaeli, R., Levi, E., Bashkin, P., Lider, O., Naparstek, Y., Cohen, I. R., and Fuks, Z. (1992). Expression of heparanase by platelets and circulating cells of the immune system: Possible involvement in diapedesis and extravasation. Invasion and Metastasis, 12, 112-127; Bartlett M. R., Underwood P. A., Parish C. R.: Comparative analysis of the ability of leukocytes, endothelial cells and platelets to degrade the subendothelial basement membrane: evidence for cytokine dependence and detection of a novel sulfatase. Immonol. Cell Biol. 1995; 73: 113-124). In these assays, detection of the products requires a synergistic activity of proteases and heparanase. Protease is required to expose HS chains to cleavage by heparanase.
Other methods involve immobilizing chemically or biosynthetically radiolabeled heparanase substrate chains (Nakajima M., Irimura T., Nicolson G. L: A solid phase substrate of heparanase: its application to assay of human melanoma for heparan sulfate degradative activity. Anal. Biochem. 1986; 157: 162-171; Oosta G. M., Favreau L. V., Beeler D. L. Rosenberg R. D: 1982. J. Biol. Chem. 257, 11249-11255; Sewell R F, Brenchley P E G, Mallick N P: Human mononuclear cells contain an endoglycosidase specific for heparan sulfate glycosaminoglycan demonstrated with the use of a specific solid-phase metabolically radiolabelled substrate. Biochem. J. 1989; 264: 777-783). The main disadvantage of these methods is that the immobilized substrate may be less accessible to the enzyme.
In the heparanase catalytic activity assay recently developed by Freeman and Parish (Freeman C, Parish C R: A rapid and quantitative assay for the detection of mammalian heparanase catalytic activity. Biochem J. 1997; 325: 229-237) the products are separated from the substrate by binding to chicken histidine-rich glycoprotein (cHRG) sepharose. In this method only the lowest molecular weight products that lose the ability to bind to cHRG sepharose are detectable, while other, longer, products bind to the column with the substrate and are therefore excluded.
The mechanism by which heparanase operates on its substrate is still unknown and it is possible that some chains may first be cleaved to longer chains and then further be degraded to smaller fragments, while other chains may be directly cleaved at the end of thereof to form small fragments. The method by Freeman and Parish, therefore, fails to detect all of the cleavage products and therefore, like all of the other prior art methods described above for assaying heparanase catalytic activity, it is qualitative rather than quantitative.
Most importantly, these heparanase activity assays are not at all adapted for automated high throughput screening.
Colorimetric heparanase assays:
PCT/US99/15643 teaches several qualitative and quantitative colorimetric assays for the detection of heparanase catalytic activity based on carbazole and dimethylmethylene blue and the detection of newly made reducing ends produced by each cleavage action of the enzyme. An inherent disadvantage to each one of these assays is that they are multiple steps assays, requiring filtration (size exclusion) steps and the like which render them inapplicable for real high throughput automated screening.
Fluorimetric heparanase assays:
Several fluorescent techniques have been developed to assay heparanase catalytic activity. These techniques are based on size exclusion separation of fluorescently labeled reaction products. For example, Toyoshima and Nakajima (Toyoshima M, Nakajima M. 1999. Human heparanase. Purification, characterization, cloning, and expression. J. Biol. Chem. 274(34):24153-60), have recently developed an assay based on high speed gel permeation chromatography of the degradation products of fluorescein isothiocyanate-labeled heparan sulfate. Partially desulfation of heparin and labeling of the resulting free amine with fluoresceinylthiocarbamoyl was previously described (Uchiyama H, Nagasawa K 1981 Preparation of biologically active fluorescent heparin composed of fluorescein-labeled species and its behavior to antithrombin III. J Biochem (Tokyo) Jan; 89(1):185-92). Reaction of 5-aminofluorescein with uronic acid residues of several glycosaminoglycuronans have yielded fluorescent glycosaminoglycuronan derivatives (Ogamo A, Matsuzaki K, Uchiyama H, Nagasawa K 1982. Preparation and properties of fluorescent glycosaminoglycuronans labeled with 5-aminofluorescein. Carbohydr Res July 1;105(1):69-85).
In a somewhat different approach, a fluorescently labeled solid phase substrate, which yields soluble labeled products upon hydrolysis, is detected following phase separation. Additional methods are labeling of either at least partially N-deacylated or N-desulfated glycosaminoglycan with (i) a substance and yielding detectable signals to produce labeled glycosaminoglycan; or, (ii) completely N-acylating the labeled glycosaminoglycan with acyl anhydride or acyl halide; or (iii) reductively aminating a reducing terminal end of said labeled glycosaminoglycan to produce labeled amine-terminal glycosaminoglycan; and (iv) coupling, through its terminal amine, the labeled amine-terminal glycosaminoglycan to an amino-reactive solid phase support to produce the solid phase substrate (U.S. Pat. Nos. 5,332,812 and 4,859,581).
Fluorescence polarization:
Fluorescence polarization was first described in 1926 (Perrin (1926) J. Phys. Rad. 1: 390-401) and has been a powerful tool in the study of molecular interactions. When fluorescent molecules are excited with plane polarized light, they emit light in the same polarized plane, provided that the molecule remains stationary throughout the excited state (e.g., 4 nanoseconds in the case of fluorescein). However, if the excited molecule rotates or tumbles out of the plane of polarized light during the excited state, then light is emitted in a different plane from that of the initial excitation. If vertically polarized light is used to excite a fluorophore, the emission light intensity can be monitored in both the original vertical plane and also the horizontal plane. The degree to which the emission intensity moves from the vertical to horizontal plane is related to the mobility of the fluorescently labeled molecule. If fluorescently labeled molecules are very large, they move very little during the excited state interval, and the emitted light remains highly polarized within the excitation plane. If the fluorescently labeled molecules are small, they rotate or tumble faster, and the emitted light is depolarized relative to the excitation plane.
Fluorescence polarization (P) is defined as:   P  =                    Int        II            -              Int        I                            Int        II            +              Int        I            
where Int(parallel) is the intensity of the emission light parallel to the excitation light plane and Int(perpendicular) is the intensity of the emission light perpendicular to the excitation light plane. P, being a ratio of light intensities, is dimensionless and has a maximum value of 0.5 for fluorescein. The Beacon System expresses polarization in millipolarization units (1 polarization Unit=1000 mP Units).
Fluorescence polarization in heparin binding assays:
Heparin has affinity to many different proteins. At one extreme the interaction between heparin and several proteins is highly specific depending on particular unusual polysaccharide sequence. Jones et al. (Jones G R, Hashim R, Power D M 1986. A comparison of the strength of binding of antithrombin III, protamine and poly(L-lysine) to heparin samples of different anticoagulant activities. Biochim Biophys Acta August 6;883(1):69-76) have shown that heparin labeled with 5-isothiocyanatofluorescein, where the dye was mostly bound to unsulphated glucosamine residues, exhibited binding which was characteristic of heparin with a low affinity for antithrombin III. On the other hand, heparin is very acidic due to its heavy substitution with sulfate groups and will bind readily to basic areas of protein surfaces in a relatively nonspecific fashion. The simplest peptides that bind to heparin are basic homopolypeptides such as poly-lysine and poly-arginine. It has long been established that heparin induces the formation of alpha-helix in these peptides (Gelman R A, Blackwell J 1973. Heparin-polypeptide interactions in aqueous solution. Arch. Biochem. Biophys. 159(1):427-33).
Using circular dichroism analysis, Mulloy et al. (Mulloy B, Crane D T, Drake A F, Davies D B 1996. The interaction between heparin and poly-lysine: a circular dichroism and molecular modeling study. Braz J Med Biol Res June; 29(6):721-9) have found that heparin oligosaccharides as small as an octasaccharide can still promote alpha-helix in poly-(L-lysine); the hexa- and tetrasaccharides do not, but they do disturb to a lesser extent the dynamic conformation equilibrium associated with poly-L-lysine at pH 7.0 at 22 degrees C. In a comparison of the strength of binding of antithrombin III, protamine and poly(L-lysine) to heparin samples of different anticoagulant activities, Jones et al. (Jones G R, Hashim R, Power D M 1986. A comparison of the strength of binding of antithrombin III, protamine and poly(L-lysine) to heparin samples of different anticoagulant activities. Biochim Biophys Acta August 6;883(1):69-76) have shown that limiting concentrations, i.e., those concentrations of sodium chloride required to completely disrupt the complexes of heparin with antithrombin III, protamine and poly(L-lysine), can be determined using fluorescence polarization techniques. They have shown that, from the limiting salt concentration values, poly(L-lysine) always exhibited stronger binding to heparin of a particular anticoagulant potency (degree of sulphation) than did protamine. The binding strengths of both complexes decreased as the degree of sulphation of the heparin participating in the complex was reduced.
The prior art, however, fails to teach a fluorescence polarization based assay for monitoring polyion (polycation or polyanion) molecular weight and changes thereof due to degradation or polymerization.
There is thus a widely recognized need for, and it would be highly advantageous to have, a fluorescence polarization based assay for monitoring polyion (polycation or polyanion) degradation or polymerization, so as to provide a high throughput assay for monitoring the activity of enzymes which degrade or polymerize polyions and for screening for potential modulators (inhibitors or activators) of such enzymes and/or to monitor physical or chemical degradation or polymerization processes of polyions, and modulators thereof. There is also a need of a fluorescence polarization based assay for determining the molecular weight of a polyion of unknown molecular weight in a sample.
While reducing the present invention to practice, an assay that utilizes the ability of NaCl to induce dissociation between heparin (a polyanion) and poly-(L-lysine) (a polycation) was developed. It was experimentally found that different limiting concentrations were required to dissociate poly-(L-lysine) and heparin of different molecular weight. This information was used to develop a potent high throughput fluorescence polarization based assay that discriminates between heparin and heparin degradation products, which assay serves as an example of the many fluorescence polarization assays provided by the present invention as is further delineated hereinbelow, all of which are based on the ability of reaction conditions, such as ionic strength, pH, temperature and/or viscosity, to induce dissociation/association between interacting polyanions and polycations in a molecular weight dependent manner.
Thus, according to the present invention there is provided a method of determining a molecular weight (e.g., an absolute or averaged molecular weight) of a first polyion in a sample, the method comprising the steps of (a) interacting the first polyion with a second polyion having an opposite charge, the second polyion being fluorescently labeled; (b) providing reaction conditions so as to allow molecular weight discriminative interaction between the first polyion and the second polyion; and (c) employing a fluorescence polarization assay for determining the molecular weight of the first polyion. Such a determination can be made using a calibration curve employing to this end samples of the first polyion of a known molecular weight.
This basic method can be used, according to the teachings of the present invention, to monitor molecular weight changes of a variety of polyions, as follows.
Hence, according to one aspect of the present invention there is provided a method of monitoring a molecular weight change of a first polyion, the method comprising the steps of (a) subjecting the first polyion to conditions under-which the first polyion undergoing the molecular weight change; (b) interacting the first polyion with a second polyion having an opposite charge, the second polyion being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the first polyion and the second polyion; and (d) employing a fluorescence polarization assay for monitoring the molecular weight change of the first polyion.
According to another aspect of the present invention there is provided a method of monitoring a molecular weight change of a polyanion, the method comprising the steps of (a) subjecting the polyanion to conditions under-which the polyanion undergoing the molecular weight change; (b) interacting the polyanion with a polycation, the polycation being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the polyanion and the polycation; and (d) employing a fluorescence polarization assay for monitoring the molecular weight change of the polyanion.
According to still another aspect of the present invention there is provided a method of monitoring a molecular weight change of a polycation, the method comprising the steps of (a) subjecting the polycation to conditions under-which the polycation undergoing the molecular weight change; (b) interacting the polycation with a polyanion, the polyanion being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the polycation and the polyanion; and (d) employing a fluorescence polarization assay for monitoring the molecular weight change of the polycation.
According to yet still another aspect of the present invention there is provided a method of monitoring degradation of a first polyion, the method comprising the steps of (a) subjecting the first polyion to degradation inducing conditions; (b) interacting the first polyion with a second polyion having an opposite charge, the second polyion being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the first polyion and the second polyion; and (d) employing a fluorescence polarization assay for monitoring a molecular weight change of the first polyion.
According to yet another aspect of the present invention there is provided a method of monitoring degradation of a polyanion, the method comprising the steps of (a) subjecting the polyanion to degradation inducing conditions; (b) interacting the polyanion with a polycation, the polycation being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the polyanion and the polycation; and (d) employing a fluorescence polarization assay for monitoring a molecular weight change of the polyanion.
According to still another aspect of the present invention there is provided a method of monitoring degradation of a polycation, the method comprising the steps of (a) subjecting the polycation to degradation inducing conditions; (b) interacting the polycation with a polyanion, the polyanion being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the polycation and the polyanion; and (d) employing a fluorescence polarization assay for monitoring a molecular weight change of the polycation.
According to yet another aspect of the present invention there is provided a method of monitoring polymerization of a first polyion, the method comprising the steps of (a) subjecting the first polyion to polymerization inducing conditions; (b) interacting the first polyion with a second polyion having an opposite charge, the second polyion being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the first polyion and the second polyion; and (d) employing a fluorescence polarization assay for monitoring a molecular weight change of the first polyion.
According to still another aspect of the present invention there is provided a method of monitoring polymerization of a polyanion, the method comprising the steps of (a) subjecting the polyanion to polymerization inducing conditions; (b) interacting the polyanion with a polycation, the polycation being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the polyanion and the polycation; and (d) employing a fluorescence polarization assay for monitoring a molecular weight change of the polyanion.
According to yet another aspect of the present invention there is provided a method of monitoring polymerization of a polycation, the method comprising the steps of (a) subjecting the polycation to polymerization inducing conditions; (b) interacting the polycation with a polyanion, the polyanion being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the polycation and the polyanion; and (d) employing a fluorescence polarization assay for monitoring a molecular weight change of the polycation.
According to still another aspect of the present invention there is provided a method of testing an agent for its potential at modulating induction of a molecular weight change of a first polyion, the method comprising the steps of (a) subjecting the first polyion to conditions under-which the first polyion undergoing the molecular weight change in a presence, in an absence or under several different concentrations of the agent; (b) interacting the first polyion with a second polyion having an opposite charge, the second polyion being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the first polyion and the second polyion; and (d) employing a fluorescence polarization assay for determining a modulating effect of the agent on the induction of the molecular weight change of the first polyion.
According to yet another aspect of the present invention there is provided a method of testing an agent for its potential at modulating induction of a molecular weight change of a polyanion, the method comprising the steps of (a) subjecting the polyanion to conditions under-which the polyanion undergoing the molecular weight change in a presence, in an absence or under several different concentrations of the agent; (b) interacting the polyanion with a polycation, the polycation being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the polyanion and the polycation; and (d) employing a fluorescence polarization assay for determining a modulating effect of the agent on the induction of the molecular weight change of the polyanion.
According to still another aspect of the present invention there is provided a method of testing an agent for its potential at modulating induction of a molecular weight change of a polycation, the method comprising the steps of (a) subjecting the polycation to conditions under-which the polycation undergoing the molecular weight change in a presence, in an absence or under several different concentrations of the agent; (b) interacting the polycation with a polyanion, the polyanion being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the polycation and the polyanion; and (d) employing a fluorescence polarization assay for determining a modulating effect of the agent on the induction of the molecular weight change of the polycation.
According to another aspect of the present invention there is provided a method of testing an agent for its potential at modulating induction of degradation of a first polyion, the method comprising the steps of (a) subjecting the first polyion to degradation inducing conditions in a presence, in an absence or under several different concentrations of the agent; (b) interacting the first polyion with a second polyion having an opposite charge, the second polyion being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the first polyion and the second polyion; and (d) employing a fluorescence polarization assay for determining a modulating effect of the agent on the induction of the molecular weight change of the first polyion.
According to yet another aspect of the present invention there is provided a method of testing an agent for its potential at modulating induction of degradation of a polyanion, the method comprising the steps of (a) subjecting the polyanion to degradation inducing conditions in a presence, in an absence or under several different concentrations of the agent; (b) interacting the polyanion with a polycation, the polycation being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the polyanion and the polycation; and (d) employing a fluorescence polarization assay for determining a modulating effect of the agent on the induction of the molecular weight change of the polyanion.
According to still another aspect of the present invention there is provided a method of testing an agent for its potential at modulating induction of degradation of a polycation, the method comprising the steps of (a) subjecting the polycation to degradation inducing conditions in a presence, in an absence or under several different concentrations of the agent; (b) interacting the polycation with a polyanion, the polyanion being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the polycation and the polyanion; and (d) employing a fluorescence polarization assay for determining a modulating effect of the agent on the induction of the molecular weight change of the polycation.
According to another aspect of the present invention there is provided a method of testing an agent for its potential at modulating induction of polymerization of a first polyion, the method comprising the steps of (a) subjecting the first polyion to polymerization inducing conditions in a presence, in an absence or under several different concentrations of the agent; (b) interacting the first polyion with a second polyion having an opposite charge, the second polyion being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the first polyion and the second polyion; and (d) employing a fluorescence polarization assay for determining a modulating effect of the agent on the induction of the molecular weight change of the first polyion.
According to yet another aspect of the present invention there is provided a method of testing an agent for its potential at modulating induction of polymerization of a polyanion, the method comprising the steps of (a) subjecting the polyanion to polymerization inducing conditions in a presence, in an absence or under several different concentrations of the agent; (b) interacting the polyanion with a polycation, the polycation being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the polyanion and the polycation; and (d) employing a fluorescence polarization assay for determining a modulating effect of the agent on the induction of the molecular weight change of the polyanion.
According to still another aspect of the present invention there is provided a method of testing an agent for its potential at modulating induction of polymerization of a polycation, the method comprising the steps of (a) subjecting the polycation to polymerization inducing conditions in a presence, in an absence or under several different concentrations of the agent; (b) interacting the polycation with a polyanion, the polyanion being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the polycation and the polyanion; and (d) employing a fluorescence polarization assay for determining a modulating effect of the agent on the induction of the molecular weight change of the polycation.
According to another aspect of the present invention there is provided a method of testing an agent for its potential at inhibiting induction of a molecular weight change of a first polyion, the method comprising the steps of (a) subjecting the first polyion to conditions under-which the first polyion undergoing the molecular weight change in a presence, in an absence or under several different concentrations of the agent; (b) interacting the first polyion with a second polyion having an opposite charge, the second polyion being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the first polyion and the second polyion; and (d) employing a fluorescence polarization assay for determining an inhibiting effect of the agent on the induction of the molecular weight change of the first polyion.
According to yet another aspect of the present invention there is provided a method of testing an agent for its potential at inhibiting induction of a molecular weight change of a polyanion, the method comprising the steps of (a) subjecting the polyanion to conditions under-which the polyanion undergoing the molecular weight change in a presence, in an absence or under several different concentrations of the agent; (b) interacting the polyanion with a polycation, the polycation being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the polyanion and the polycation; and (d) employing a fluorescence polarization assay for determining an inhibiting effect of the agent on the induction of the molecular weight change of the polyanion.
According to still another aspect of the present invention there is provided a method of testing an agent for its potential at inhibiting induction of a molecular weight change of a polycation, the method comprising the steps of (a) subjecting the polycation to conditions under-which the polycation undergoing the molecular weight change in a presence, in an absence or under several different concentrations of the agent; (b) interacting the polycation with a polyanion, the polyanion being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the polycation and the polyanion; and (d) employing a fluorescence polarization assay for determining an inhibiting effect of the agent on the induction of the molecular weight change of the polycation.
According to another aspect of the present invention there is provided a method of testing an agent for its potential at inhibiting induction of degradation of a first polyion, the method comprising the steps of (a) subjecting the first polyion to degradation inducing conditions in a presence, in an absence or under several different concentrations of the agent; (b) interacting the first polyion with a second polyion having an opposite charge, the second polyion being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the first polyion and the second polyion; and (d) employing a fluorescence polarization assay for determining an inhibiting effect of the agent on the induction of the molecular weight change of the first polyion.
According to yet another aspect of the present invention there is provided a method of testing an agent for its potential at inhibiting induction of degradation of a polyanion, the method comprising the steps of (a) subjecting the polyanion to degradation inducing conditions in a presence, in an absence or under several different concentrations of the agent; (b) interacting the polyanion with a polycation, the polycation being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the polyanion and the polycation; and (d) employing a fluorescence polarization assay for determining an inhibiting effect of the agent on the induction of the molecular weight change of the polyanion.
According to still another aspect of the present invention there is provided a method of testing an agent for its potential at inhibiting induction of degradation of a polycation, the method comprising the steps of (a) subjecting the polycation to degradation inducing conditions in a presence, in an absence or under several different concentrations of the agent; (b) interacting the polycation with a polyanion, the polyanion being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the polycation and the polyanion; and (d) employing a fluorescence polarization assay for determining an inhibiting effect of the agent on the induction of the molecular weight change of the polycation.
According to another aspect of the present invention there is provided a method of testing an agent for its potential at inhibiting induction of polymerization of a first polyion, the method comprising the steps of (a) subjecting the first polyion to polymerization inducing conditions in a presence, in an absence or under several different concentrations of the agent; (b) interacting the first polyion with a second polyion having an opposite charge, the second polyion being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the first polyion and the second polyion; and (d) employing a fluorescence polarization assay for determining an inhibiting effect of the agent on the induction of the molecular weight change of the first polyion.
According to yet another aspect of the present invention there is provided a method of testing an agent for its potential at inhibiting induction of polymerization of a polyanion, the method comprising the steps of (a) subjecting the polyanion to polymerization inducing conditions in a presence, in an absence or under several different concentrations of the agent; (b) interacting the polyanion with a polycation, the polycation being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the polyanion and the polycation; and (d) employing a fluorescence polarization assay for determining an inhibiting effect of the agent on the induction of the molecular weight change of the polyanion.
According to still another aspect of the present invention there is provided a method of testing an agent for its potential at inhibiting induction of polymerization of a polycation, the method comprising the steps of (a) subjecting the polycation to polymerization inducing conditions in a presence, in an absence or under several different concentrations of the agent; (b) interacting the polycation with a polyanion, the polyanion being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the polycation and the polyanion; and (d) employing a fluorescence polarization assay for determining an inhibiting effect of the agent on the induction of the molecular weight change of the polycation.
According to another aspect of the present invention there is provided a method of testing an agent for its potential at activating induction of a molecular weight change of a first polyion, the method comprising the steps of (a) subjecting the first polyion to conditions under-which the first polyion undergoing the molecular weight change in a presence, in an absence or under several different concentrations of the agent; (b) interacting the first polyion with a second polyion having an opposite charge, the second polyion being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the first polyion and the second polyion; and (d) employing a fluorescence polarization assay for determining an activating effect of the agent on the induction of the molecular weight change of the first polyion.
According to yet another aspect of the present invention there is provided a method of testing an agent for its potential at activating induction of a molecular weight change of a polyanion, the method comprising the steps of (a) subjecting the polyanion to conditions under-which the polyanion undergoing the molecular weight change in a presence, in an absence or under several different concentrations of the agent; (b) interacting the polyanion with a polycation, the polycation being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the polyanion and the polycation; and (d) employing a fluorescence polarization assay for determining an activating effect of the agent on the induction of the molecular weight change of the polyanion.
According to still another aspect of the present invention there is provided a method of testing an agent for its potential at activating induction of a molecular weight change of a polycation, the method comprising the steps of (a) subjecting the polycation to conditions under-which the polycation undergoing the molecular weight change in a presence, in an absence or under several different concentrations of the agent; (b) interacting the polycation with a polyanion, the polyanion being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the polycation and the polyanion; and (d) employing a fluorescence polarization assay for determining an activating effect of the agent on the induction of the molecular weight change of the polycation.
According to another aspect of the present invention there is provided a method of testing an agent for its potential at activating induction of degradation of a first polyion, the method comprising the steps of (a) subjecting the first polyion to degradation inducing conditions in a presence, in an absence or under several different concentrations of the agent; (b) interacting the first polyion with a second polyion having an opposite charge, the second polyion being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the first polyion and the second polyion; and (d) employing a fluorescence polarization assay for determining an activating effect of the agent on the induction of the molecular weight change of the first polyion.
According to yet another aspect of the present invention there is provided a method of testing an agent for its potential at activating induction of degradation of a polyanion, the method comprising the steps of (a) subjecting the polyanion to degradation inducing conditions in a presence, in an absence or under several different concentrations of the agent; (b) interacting the polyanion with a polycation, the polycation being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the polyanion and the polycation; and (d) employing a fluorescence polarization assay for determining an activating effect of the agent on the induction of the molecular weight change of the polyanion.
According to still another aspect of the present invention there is provided a method of testing an agent for its potential at activating induction of degradation of a polycation, the method comprising the steps of (a) subjecting the polycation to degradation inducing conditions in a presence, in an absence or under several different concentrations of the agent; (b) interacting the polycation with a polyanion, the polyanion being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the polycation and the polyanion; and (d) employing a fluorescence polarization assay for determining an activating effect of the agent on the induction of the molecular weight change of the polycation.
According to another aspect of the present invention there is provided a method of testing an agent for its potential at activating induction of polymerization of a first polyion, the method comprising the steps of (a) subjecting the first polyion to polymerization inducing conditions in a presence, in an absence or under several different concentrations of the agent; (b) interacting the first polyion with a second polyion having an opposite charge, the second polyion being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the first polyion and the second polyion; and (d) employing a fluorescence polarization assay for determining an activating effect of the agent on the induction of the molecular weight change of the first polyion.
According to yet another aspect of the present invention there is provided a method of testing an agent for its potential at activating induction of polymerization of a polyanion, the method comprising the steps of (a) subjecting the polyanion to polymerization inducing conditions in a presence, in an absence or under several different concentrations of the agent; (b) interacting the polyanion with a polycation, the polycation being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the polyanion and the polycation; and (d) employing a fluorescence polarization assay for determining an activating effect of the agent on the induction of the molecular weight change of the polyanion.
According to still another aspect of the present invention there is provided a method of testing an agent for its potential at activating induction of polymerization of a polycation, the method comprising the steps of (a) subjecting the polycation to polymerization inducing conditions in a presence, in an absence or under several different concentrations of the agent; (b) interacting the polycation with a polyanion, the polyanion being fluorescently labeled; (c) providing reaction conditions so as to allow molecular weight discriminative interaction between the polycation and the polyanion; and (d) employing a fluorescence polarization assay for determining an activating effect of the agent on the induction of the molecular weight change of the polycation.
The present invention successfully addresses the shortcomings of the presently known configurations by providing a high throughput fluorescence polarization assay for monitoring degradation or polymerization of polyanions or polycations, which can be used to screen for modulators (inhibitors and activators) of these processes.